This invention relates to methods for modifying polyhydroxylated materials by covalently bonding nucleophilic ligands to said material. More specifically, this invention relates to improved support materials for use as stationary phases in various chromatographic methods, and to methods for preparing such support materials.
Chromatography is a separation technique whereby individual chemical compounds which were originally present in a mixture are resolved from each other by the selective process of distribution between two heterogeneous (immiscible) phases. The distribution of chemical species to be separated occurs in a dynamic process between a mobile phase and a stationary phase. The stationary phase is a dispersed medium, which usually has a relatively large surface area, through which the mobile phase is allowed to flow. The chemical nature of the stationary phase exercises the primary control over the separation process. The greater the affinity of a particular chemical compound (referred to as the solute) for the stationary medium, the longer it will be retained in the system. The mobile phase can be either gas or liquid; correspondingly, the methods are referred to as gas chromatography and liquid chromatography.
There are a wide variety of chromatographic methods, varying, for example, in the selection of mobile and stationary phases, techniques and solute measurement principles. As an example, ion exchange chromatography is a widely used form of liquid chromatography. It is based on selective ionic attractions between variously charged sample constituents and an ionized chromatographic matrix. The most commonly used ion exchangers consist of an organic polymeric backbone with either acidic or basic exchange sites on its porous surface. The charged resins are capable of exchanging their cations or anions with those ions in the liquid phase which have a greater affinity for the matrix. Exchange interactions that take place during the passage of various ions through the column cause separation into discrete ionic zones.
Thin layer chromatography is a technique in which the stationary phase is a suspension which forms a layer on a plastic or glass plate. It is most frequently an adsorbent (with a particle size of several microns) suspended in a suitable solvent, uniformly spread on a plate, and dried. The mobile phase is a liquid that ascends the plate by capillary action, and the components of the sample mixture are separated by the partition effect.
Reverse-phase chromatography is a type of chromatography in which hydrocarbons as well as polar samples are partitioned between a nonpolar stationary phase and a polar eluting phase. Under these conditions the most polar substances elute most rapidly. This is the reverse of the more common partition chromatography in which the stationary phase is polar and the least polar substances elute most rapidly with the nonpolar eluting phase. In reverse phase chromatography, the stationary phase often consists of a chain of atoms chemically bonded to an inert surface such as silica or glass, and the eluting phase is frequently aqueous methanol or aqueous acetonitrile.
Molecular sieve chromatography, often called gel chromatography, has resulted in tremendous progress in the chemistry of biomacromolecules. Separation in molecular sieve chromatography is based on a selective process of penetration of molecules of different sizes and shapes through a porous gel medium. The largest molecules in the mixture do not penetrate the porous structure at all; the medium-size molecules can penetrate only some pores; and the small molecules can diffuse rather freely inside the medium and can spend a considerably longer time there. Consequently, if the porous material is contained in a column, mixtures of components with differing molecular weights can be effectively resolved.
In any chromatographic process, some components of a given mixture will be retained on the stationary phase longer than others. This allows for extremely selective chromatographic separations. For example, in the method called affinity chromatography, molecules to be purified interact with immobilized ligands on the surface of the stationary phase and are strongly retained by the stationary phase material Passage of a multicomponent extract through a column of immobilized ligand results in selective adsorption of the recognized material to the column. Non-interacting material can be washed away, and bound components can be eluted biospecifically with competetive or affinity modifying reagents, or under denaturing conditions. It is therefore always necessary to seek a stationary phase material with a selectivity appropriate to a given separation problem.
Common to all of the above-described chromatography methods is the use of a stationary phase having at its surface a phase which will interact with the desired components of the mobile phase in the desired manner, e.g., the highly specific ligands attached to the stationary phase in affinity chromatography, or the acidic or basic exchange sites on the stationary phase in ion exchange chromatography. Development of stationary phases for various types of chromatography in general has focused on the attachment of various bonded phases to dextran (Sephadex), agarose, glass, silica and polymeric materials such as polyacrylamide, polymethacrylates or latex.
More specifically, for use in affinity chromatography, the chemistry of ligand immobilization using activation of agarose with cyanogen bromide has been the most popular methodology. The generation of carbonates and caoamates by reaction with 1,1'-carbonyldiimidazole or chloroformate with agarose, polyacrylamide, cellulose, glass beads or hydroxylated polystyrene or other polymers has also been employed. A major disadvantage of such chemistries is the production, on reaction with amines, of a relatively unstable amide bond resulting in continuous ligand leakage at a slow but measureabIe rate. Moreover, ionic contributions to non-specific protein adsorption are also observed, probably due to the formation of isourea groups. The use of carbonyl diimidizole activated supports which on reaction with amines form a urethane linxage, as well as use of bifunctional oxiranes, has reduced but not eliminated non-specific protein binding. Despite these improvements, agarose remains susceptible to microbial attack, is of limited usefulness in the presence of organic solvents, and is not amenable to easy scale-up and high flow rates. Other supports such as glass, while performing well in organic solvents, suffer from residual charged functions and non-specific binding. Polyacrylamide, while more resistant to microbial attack an agarose, does not form high flow capacity columns. Other polymeric material suffer from higher levels of non-specific interactions than agarose.
Ideally, stationary phases, or chromatography supports, should have good mechanical strength and flow properties, be available in a range of particle sizes, pore sizes and shapes, be chemically stable, possess a high level of hydrophilicity, be amenable to a number of modifications and possess little or no non-specific interaction with the components to be resolved. Silica has been shown to satisfy most of these criteria. Optical activation and performance of silica columns for use in affinity chromatography has been achieved with spherical 10 micron particles. No significant advantage was obtained with smaller particles, and substantial decreases in performance were observed with 20 micron materials. Important advantages accruing from the use of silica supports in affinity chromatography have been found, including high accessible capacity, complete resistance to microbial attack, ease and versatility of immobilization chemistry, high purification efficiency and excellent flow properties. See Hollis et al., J. Liq. Chromat., 10, 2349 (1987). Use of silica results in affinity chromatography systems where elution volumes are minimized and procedures are rapid and easily automated.
Previously, silica has been modified for use in chromatography via a series of reactions using various organosilane analogues and methodologies. These chemistries result in bonded phase attachment via an Si--O--Si linkage which is sensitive and labile to acid, base and other treatments. A generalized scheme representing silane activation of silica is a follows: ##STR1## As shown in Scheme 1, the reactive organo silane is directed towards available hydroxyl functions on the silica surface. The half-life of such supports are variable and unpredictable due to slow decomposition of the Si--O--Si bond. In particular, silane activated silicas are unstable above a pH of about 7.2. While this has not hindered the use of silica for applications requiring a pH in the range of 2-7.2, different supports such as polymeric beads must be used for applications requiring a higher pH. Many polymeric beads are stable at elevated pH but show poor flow characteristics and higher non-specific binding when compared to silica. In some cases, zirconium impregnated or polymer coated silicas have been prepared and are claimed to possess higher pH stability.
Thus, although silica provides a chromatographic support material which is advantageous in comparison to other materials in many respects, chemical instability stemming from the chemical approach of attaching a ligand is a severe drawback. There are many separation applications which would benefit from the ability to use a silica support having greater pH stability, e.g., biochromatographic methods involving the separation of proteins, DNA, RNA, cells or cellular particles in a format designed to maintain any associated biological activity. Examples of such biochromatographic techniques are ion exchange, hydrophobic interaction, size exclusion separations, as well as the above-described affinity chromatography. Because of the chemical instability of surface-modified silica, and because optimum bio-separations are usually observed at an alkaline pH, many such biochromatographic separations employ polymer packings possessing less efficient flow properties and substantially greater non-specific interactions than silica.
There is thus a need for methods for preparing silica supports in the absence of impregnated or polymer coating materials which would display little or no non-specific interaction with components to be resolved and which would maintain the excellent flow properties of silica. In addition, there is a need for methods for preparing silica supports which would have greater pH stability than the standard organo silane-activated silicas in use today.
Towards this end, we hypothesized that a silica particle, with its numerous hydroxyl functions, could be viewed as a "polyol" wherein some, if not all, hydroxyls are assumed to be in close proximity and in cis-configuratoin, i.e., as one encounters in a sugar molecule. It is known that a nucleophile may be introduced to a 1,2-cis-diol, such as ribose, by application of carbohydrate phosphate chemistry as illustrated in Scheme 2. ##STR2## Thus, we hypothesized that a nucleophile could be introduced to a silica surface using the same general chemistry, as illustrated in Scheme 3. ##STR3##
Tests have indicated that such chemistry can indeed be utilized to introduce nucleophiles suitable for chromatography on the surface of silica. Advantageously, in the materials prepared in this manner, the nucleophiles are covalently bound directly to a silicon atom of the backbone, yielding a Si-Nu linkage (Scheme 3) which is more stable than the acid-base-sensitive Si--O--Si--Nu linkage (Scheme 1) arising in standard organo silane-activated silicas, thus avoiding one of the major problems inherent in the use of silica chromatography supports to date. This work has implications far broader, however, than only the modification of silica materials. If it can be assumed that at least a portion of the hydroxyl functions on the surface of a silica support are in close proximity and in cis-configuration, and the work described herein bears out this assumption, then it can also be assumed that at least a portion of the hydroxyl functions on the surface of other polyhydroxylated support materials would also be so arranged. Thus, the activation/nucleophilic displacement chemistry described above can be extended to include the modification of a wide variety of polyhydroxylated materials, exemplified by those described in this patent, in a variety of ways.